Activity-dependent regulation of neuronal function is initiated in large part via depolarization-induced calcium influx. Neuronal calcium has been shown to activate a number of intracellular kinase cascades to regulate processes such as gene transcription, synaptic plasticity and cell survival. Many of these activity-dependent functions are shared by the neurotrophin family of growth factors suggesting a common signaling pathway. Two pathways, the cAMP independent protein kinase (PKA) cascade and the extracellular signal regulated kinase (ERK) cascade have been implicated as potential targets for neuronal calcium-mediated signaling. Recent data in the applicant's laboratory have identified a novel signaling pathway downstream of NGF and cAMP signaling. This pathway involves PKA-dependent activation of the Ras-related small G protein, Rap1, and subsequent activation of a neuronal-specific ERK cascade. The major hypothesis of this proposal is that stimulation of this Rap-dependent ERK cascade is a target for activity-dependent calcium influx. This proposal utilizes membrane depolarization of both the PC12 neuronal cell line and hippocampal neurons to mimic activity-dependent regulation of calcium influx. The applicant will test the hypothesis that the small G protein Rap1 participates in the depolarization-mediated activation of ERKs. Recent studies have shown that neuronal Rap1 is activated by multiple second messengers. Therefore, the applicant further proposes to examine the possibility that, like neurotrophic signaling, PKA is required for neuronal calcium activation of Rap1. Finally, the applicant will also test the hypothesis that activation of Rap1 can couple calcium influx to two nuclear actions of ERKs: the nuclear translocation of ERKs themselves and CREB-dependent transcription. The work outlined in this grant will impact many areas of neuroscience and will point to Rap1 as a confluence point of neuronal signals. Given the well documented role for PKA and CREB in these long-term changes that accompany neuronal activity, it is likely that these studies in PC12 cells will identify the importance of Rap-dependent pathways in the long-term changes. The applicant believes that the experiments outlined in this proposal may ultimately impact more general research on synaptic plasticity, long-term potentiation (LTP) and neuronal survival.